Natural Killer T cells (NKT) are a hybrid cell type of NK cells and T cells, but are activated only by antigenic stimulation. NKT cells bridge innate and adaptive immunity through recognition of lipid ligands from both microbial and self origins. Endogenous NKT ligands, which are metabolism products of self lipids, are involved in immuno-pathology of infectious diseases, auto-immune diseases and cancer. However, molecular identification of these endogenous ligands has been difficult due to technical barriers. Recently, we found that NB-DGJ, a drug which inhibits glucosylceramide synthase, completely abolishes the development of NKT cells. More importantly, we identified isoglobotrihexosylceramide, one of the target glycosphingolipids being inhibited by NB-DGJ, as the first known natural ligand for NKT cells. However, iGb3 synthase knockout mice showed no defect in NKT cell development and function, indicating that other GSL and/or non-GSL ligands exist. Continued development of glycolipidomics assays, assisted by ion trap mass spectrometry technology, enhances our ability to further understand these mechanisms and identify new ligands. In this project, we will test the hypothesis that at least one endogenous ligand of NKT cells, other than iGb3, exists and is responsible for the development and activation of NKT cells. Identifying one or more endogenous ligands may permit new approaches to regulating the function of NKT cells in disease settings such as cancer and autoimmune diseases. The specific aims of this project are: 1) Determine the identities of stimulatory GSL and/or non-GSL antigens by biochemical fractionation and analysis; 2) Utilize the genetic pathways to identify potential endogenous NKT ligands. The molecular identification of natural ligands for NKT cells is not only an urgent, but also a challenging task in the field of innate immunity. Although the search for these ligands has lasted more than a decade, they remain elusive. We therefore have chosen a combined approach of biochemical fractionation/characterization and genetic knockdown of key enzymes to help bring this search to fruition. The completion of human genome project and the advancement of mass spectrometry technology provide the framework to move the field of lipid antigens forward and potentially permit the identification of new therapeutic approaches to some of the most complex immune-related diseases. More important, this project will provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycolipids. More than 10% of the genes in the human genome are used for lipid metabolism; thus, knowledge and experience gained in this project may have profound implications on studies of functional lipidomics, an important but little-understood area compared with proteomics and genomics.